* Retinoblastoma is the most common primary intraocular tumor of
childhood and may be heritable or occur sporadically. Anterior diffuse
retinoblastoma is an uncommon variant that is thought to be sporadic. We
describe a child with anterior diffuse retinoblastoma who presented with
a pseudohypopyon. Genetic analysis showed a germline mutation of the RB1
allele that is potentially heritable. Immunofluorescence staining was
positive for transforming growth factor P and for vascular endothelial
growth factor and negative for inducible nitric oxide synthase and for
hypoxia inducible factor a in the tumor seeds, indicating acquisition of
nonischemia-mediated survival factors of the tumor seeds in the aqueous
humor.

Retinoblastoma is the most common primary intraocular tumor in
childhood. When a family history is present, 50% of offspring of a
parent carrying an RB1 mutant allele will inherit that allele; 40% of
them will develop retinoblastoma tumors. Each tumor that forms will have
lost the second RB1 allele. Diffuse anterior retinoblastoma presents in
older children and may mimic uveitis because it presents as cells in the
posterior compartment and anterior chamber without an obvious retinal
mass. Diffuse anterior retinoblastoma has previously been described as
occurring in sporadic mutations. Herein, we report on a patient with
diffuse anterior retinoblastoma who is the first member of a family to
carry a germline RB1 mutant allele. Immunofluorescence staining showed
that tumor seeds in the aqueous humor and implanted in the iris
expressed nonischemia-related survival factors transforming growth
factor p (TGRp) and vascular endothelial growth factor (VEGF).

REPORT OF A CASE

A 9-year-old girl was evaluated for blurry vision in her left eye.
The patient was otherwise healthy, and there was no family history of
eye disease. The patient's mother had noted redness and changes in
color of the left eye during the preceding 2 months. Examination showed
20/20 vision in the right eye and 20/60 vision in the left eye. The
intraocular pressures were 16 and 34 mm Hg in the right and left eyes,
respectively. Examination showed a normal right eye. Anterior segment
examination showed a pseudohypopyon in the left eye (Figure 1). Dilated
fundus examination showed a possible small, inferior, peripheral mass in
her left retina. An anterior-chamber fine-needle aspiration biopsy
showed a small, round blue cell tumor, consistent with retinoblastoma
(Figure 2, A and B). Complete blood cell count and bone marrow biopsy
were performed, and findings from both were normal. The left eye was
enucleated. Mutational analysis of the patient's tumor showed 2 RB1
mutant alleles: a nonsense mutation c.763C [right arrow] T(R255X) and a
deletion, c.1572delA. The nonsense mutation was also identified in the
DNA from the patient's blood, indicating a germline mutation
(Table).

PATHOLOGIC FINDINGS

Gross examination of the enucleated eye showed white material in
the anterior chamber. Similar white material was present anterior to the
vitreous base and extended circumferentially for 360[degrees] (Figure 3,
A). A small, white tumor was present in the peripheral retina near the
ora serrata inferiorly. Scattered retinal hemorrhages with white centers
were present (Figure 3, B and C). Microscopic examination showed
fibrovascular tissue on the anterior surfaces of the iris leaflets
causing ectropion uveae. The tumor was present in the anterior chamber
and implanted into the iris (Figure 4, A). Islands of tumor and
individual tumor cells were present in the space between the vitreous
base and ciliary body (Figure 4, B). Tumor cells extended within the
aqueous humor, through the pupil, around the iris, and into the anterior
chamber. There were occasional Homer Wright rosettes in the tumor,
especially in the islands of tumor in the anterior chamber. The
peripheral inferior retina contained a 3 X 1 mm focus of tumor. The
tumor was composed of small, round blue cells with high nuclear to
cytoplasmic ratios and hyperchromatic nuclei. There was a hemorrhage
associated with the intraretinal tumor. The tumor was confined to the
retina and overlying aqueous by the anterior hyaloid face of the
vitreous by a tamponade effect of the vitreous humor (Figure 4, C). The
tumor did not invade into the subretinal pigment epithelium space or
into the choroid. Immunofluorescent staining for TGF-[beta] 1/2/3 (Santa
Cruz Biotechnology, Santa Cruz, California), VEGF (Santa Cruz
Biotechnology), inducible nitric oxide synthase (iNOS; R&D Systems,
Minneapolis, Minnesota), and hypoxia inducible factor (HIF; Santa Cruz
Biotechnology) was performed. Tumor seeds in the aqueous humor stained
positively for TGF-[beta] and VEGF (Figure 5, A through D). The retinal
focus stained for VEGF alone. The tumor seeds and intraretinal tumor
failed to stain for iNOS or HIF1a.

[FIGURE 1 OMITTED]

COMMENT

Growth patterns of retinoblastoma can be endophytic, exophytic, or
diffuse. Most retinoblastomas exhibit both exophytic and endophytic
components. Anterior diffuse retinoblastoma, a variant of diffuse
retinoblastoma, results in 360[degrees] seeding in the area of the
vitreous base/ciliary body with an associated anterior chamber
pseudohypopyon. (1-3) The diffuse growth pattern results in late
clinical diagnoses because there is no retinal mass, and associated
tumor seeding may mimic uveitis. Children with diffuse infiltrating
retinoblastoma are usually diagnosed between ages 5 and 12 years, with a
mean age at diagnosis of 6.1 years. (4) One case of diffuse
retinoblastoma showed loss of heterozygosity in diffuse retinoblastoma
cells. (5) Thereisan inconspicuous, peripheral focus of intraretinal
retinoblastoma in anterior diffuse retinoblastoma, which results in
shedding of tumor cells into the aqueous humor between the ciliary
epithelium and vitreous base, with the aqueous humor carrying the tumor
into the anterior chamber. This form of retinoblastoma is unilateral and
has been thought to be nonheritable. Our case exhibited a germline
mutation that has been previously reported in heritable retinoblastoma,
(6) although it is unclear whether that case was an isolated unilateral
tumor or a multifocal and bilateral tumor.

To better understand the cell signaling and survival mechanisms in
this unusual variant of retinoblastoma, we performed immunofluorescent
staining for TGF-[beta], VEGF, iNOS, and HIF. Tumor growth factor p
exhibits tumor suppression via effects on proliferation, replication
potential, and apoptosis. Tumor growth factor [beta] also exhibits tumor
promotion via effects on migration, invasion, angiogenesis, and the
immune system. The dual nature of TGF-[beta] is dependent on the cell
type and genetic status of proteins in the signal transduction pathway.
(7) Interestingly, RB1 can regulate TGF-[beta] gene expression via
cyclin-dependent kinase inhibitors, depending on the cell type. (8)
Vascular endothelial growth factor is a member of the tyrosine kinase
platelet-derived growth factor superfamily and includes placental growth
factor, VEGF-A through VEGF-E. Vascular endothelial growth factor has
been mapped to 6pter-p21 and is a multifunctional cytokine involved with
angiogenesis, vasculogenesis, and cell survival. (9) Inducible nitric
oxide synthase generates nitric oxide from L-arginine, modulates
malignant transformation, angiogenesis, metastasis; mediates
angiogenesis via VEGF and basic fibroblast growth factor; and is
expressed in ischemic retina, where it mediates intraretinal to
intravitreal angiogenesis. (10,11) Hypoxia inducible factor 1 is the
primary mediator of low oxygen-tension environments (hypoxia) leading to
angiogenesis. (12) The tumor seeds in the aqueous humor in our case
expressed TGF-[beta] and VEGF and did not express iNOS or HIF1. The
intraretinal tumor expressed VEGF, which did not appear to be mediated
by HIF1 or iNOS, the most common pathways of ischemia mediated
angiogenesis. We speculate that the tumor expression of VEGF was likely
not due to ischemia, and tumor seeds acquired the expression of
TGF-[beta], a survival factor, in the aqueous humor.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Anterior diffuse retinoblastoma is a variant of diffuse
retinoblastoma that arises as a small focus in the peripheral retina.
Free tumor cells express TGF-[beta] and VEGF, are spread via the aqueous
humor between the vitreous base and ciliary epithelium for 360[degrees],
migrate through the pupil where they become implanted into the iris, and
cause neo vascularization of the iris. This is a microcosm of neoplastic
transformation, wherein tumors acquire the ability for limitless
replication, evasion of apoptosis, and production of self-sufficient
growth signals; become insensitive to antigrowth signals; produce
sustained angiogenesis; and invade tissue via metastasis. (13) In our
case, there was a germline mutation, indicating that this form of
retinoblastoma may be heritable.

[FIGURE 5 OMITTED]

This study was supported, in part, by grant EY06360 from the
National Institutes of Health, and an unrestricted departmental grant
from Research to Prevent Blindness, Inc. Several pictures and technical
details were acquired with the significant aid of Weiqing Gao, BS. We
are deeply grateful for her assistance in the preparation of this
manuscript.